You clone the GFP cDNA into the Pst I and Xho I cloning sites of a plasmid vecto

Question

You clone the GFP cDNA into the Pst I and Xho I cloning sites of a plasmid vector, containing an ampicillin selective marker. You transform the vector and select several colonies that grew on LB plates containing ampicillin. You isolate the plasmid and do a double digest with Pst I and Xho I restriction enzymes. After running the products on a gel you confirm you have a potential clone. After DNA sequencing everything looks fine, all samples contained the clone and the DNA sequence showed no mistakes. when you go to express your protein you find that absolutely no protein is being made, but the cells are still growing. What is your explanation for this?

Explanation / Answer

The protein expression can be detrimental to various factors. Essentially, GFP cDNA encoding a target protein is cloned downstream into the Pst I and Xho I cloning sites of a promoter in an expression vector.

This vector is then introduced by transformation technique into a host cell, under an assumption that cell’s protein synthesis machinery produces the desired protein. In practice, however, protein expression can be very challenging because so many factors may influence the process.

Although everything looks perfect in sequencing there could be intrisic factors that affect protein expression. For instance, each protein folds in its own unique manner, a process that may be influenced by the choice of expression host.

Similarly, some proteins need post-translational alterations or proper insertion into a biological membrane. On the other hand, some proteins may have an movement that is detrimental to the host although not the case here.

Thus, no single answer exists for successful manufacture of all recombinant proteins. Instead, it is promising to have access to a wide range of expression tools, and a desire to explore multiple approaches to foster one’s chances for success.

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